Probiotic Lactobacillus plantarum Ln4 Showing Antimicrobial and Antibiofilm Effect against Streptococcus mutans KCTC 5124 Causing Dental Caries

Dental caries has known as an infectious disease that is considered a serious global public health problem. Recently, report indicate that probiotics play a vital role in maintaining oral health. Therefore, this study aimed to evaluate the prevention effects of Lactobacillus plantarum Ln4 against dental infection by the pathogenic bacterium Streptococcus mutans KCTC 5124 through biofilm formation inhibition. To evaluate such prevention effects against S. mutans KCTC 5124, antimicrobial activity, auto-aggregation, co-aggregation, cell surface hydrophobicity, total exopolysaccharide (EPS) production rate, and biofilm formation were analyzed. Results showed that L. plantarum Ln4 showed higher antimicrobial activity than L. rhamnosus GG (LGG). In the group treated with L. plantarum Ln4, the co-aggregation (58.85%), cell surface hydrophobicity (16.75%), and EPS production rate (73.29%) values were lower than those of LGG and the negative control. Additionally, crystal violet staining and confocal laser scanning microscopy (CLSM) revealed that L. plantarum Ln4 effectively inhibited biofilm formation in S. mutans KCTC 5124. Therefore, L. plantarum Ln4 could be used in the industry as a probiotics to prevent and improve oral health.


Hydrophobicity Determination
The hydrophobicity of S. mutans KCTC 5124 treated Lactobacillus strains was determined as previously described, with some modifications [22,24].Xylene was used to characterize hydrophobicity.Briefly, culture strains were incubated in MRS broth at 37°C for 24 h and centrifuged at 14,240 ×g for 5 min; culture supernatants were washed twice and resuspended in PBS buffer.Next, resuspended cells were adjusted to an OD 600 of 0.5 (OD Initial ).Resuspended cells (3 ml) were then added to 1 ml of solvent (xylene), pre-incubated at 37°C for 10 min in an incubator, and incubated at 37°C for 20 min.After incubation, the mixture was separated into two phases.The aqueous phase (1 ml) was collected, and the its absorbance was measured at 600 nm (OD Time ). Hydrophobicity was calculated as follows: Cell surface hydrophobicity (%) = (1 -(OD Time / OD Initial )) × 100

Total Exopolysaccharide (EPS) Production Rate
The total EPS production rate was evaluated as previously described, with some modifications [23].Briefly, Lactobacillus strains and S. mutans KCTC 5124 were grown in MRS broth and BHI broth with 3% sucrose at 37°C for 24 h in a 5% CO 2 incubator.S. mutans KCTC 5124 was mixed with the MIC of Lactobacillus and LGG strains in BHI broth containing 3% sucrose at 37°C for 24 h in an anaerobic incubator.The suspensions were centrifuged at 8,000 ×g for 10 min, and the supernatants were collected.Supernatant samples (1 ml) were added to 2 ml of 99% ethyl alcohol and incubated at 4°C for 24 h.The mixtures were centrifuged at 14,240 ×g for 15 min, and the pellets were resuspended by using distilled water (500 μl).Cell suspensions (40 μl) were combined with 40 μl of 5% phenol and 4 ml of 95% sulfuric acid, and the reaction was carried out for 10 min at 30°C.Absorbance was measured to determine the total EPS production rate, which was calculated as follows: EPS production rate (%) = (OD Treatment / OD Control ) × 100

Biofilm Formation Using Crystal Violet Staining
Biofilm formation was evaluated using the method reported by Lim et al. [8] with some modifications.Briefly, S. mutans KCTC 5124 was seeded on a 24-well plate (1 × 10 6 CFU/ml) and incubated at 37°C for 48 h in an anaerobic incubator.Each well was then inoculated with the supernatant of Lactobacillus strains, or LGG at a concentration of 10 9 CFU/ml (100 μl, 200 μl, and 500 μl per well) and incubated at 37°C for 15 h in a 5% CO 2 incubator.After incubation is over, the planktonic mixtures were removed and washed three times with PBS buffer.The plates were dried at 37°C for 10 min, stained using 0.1% crystal violet solution for 10 min.Next, the stained plates were washed, rinsed with distilled water, and air-dried completely at room temperature.A solvent mixture (10% acetic acid, 30% methanol, and 60% distilled water) was added to each well and shaken until crystal violet dissolved.Absorbance was measured at 570 nm, and the biofilm inhibition rate was calculated as follows: Biofilm inhibition rate (%) = (1 -(OD Sample / OD Control )) × 100

Confocal Laser Scanning Microscopy (CLSM)
CLSM was conducted to quantitatively evaluate the inhibition of S. mutans KCTC 5124 biofilm formation by Lactobacillus strains, with modifications [23].Briefly, S. mutans KCTC 5124 was cultured in BHI broth containing 3% sucrose at 37°C for 24 h in a 5% CO 2 incubator, and Lactobacillus and LGG strains were cultured in MRS broth at 37°C for 24 h.S. mutans KCTC 5124 was seeded in a 6-well plate (1 × 10 6 CFU/ml), and glass coupons were added.Lactobacillus and LGG strains were treated with MIC concentration, while the control group was treated with BHI broth containing 3% sucrose.After incubation at 37°C for 24 h, glass coupons were washed with PBS buffer and stained with 1 μM/ml of SYTO9 (Invitrogen, Thermo Fisher Scientific, USA) for 20 min in the dark at room temperature.Glass coupons were then washed twice with PBS buffer and air-dried for 40 min in the dark.Glass coupons were fixed with coverslips and observed using a confocal laser scanning microscopy (Carl Zeiss, Germany).

Statistical Analysis
Results for each treatment were obtained in triplicate, and one-way analysis of variance (SPSS software version 19; IBM, USA) and Student's t-test were performed to determine the significance of the differences among the mean values.Results are presented as the mean ± standard deviation.

Antimicrobial Effect against S. mutans KCTC 5124
The antimicrobial activity of Lactobacillus strains against S. mutans is shown in Table 1.In general, L. plantarum strains exhibited better antimicrobial activity than LGG.Particularly, L. plantarum Ln4 showed a large clear zone (30.33 mm) against oral pathogenic S. mutans KCTC 5124 (p < 0.05).Although LGG showed the largest clear zone (31.33 mm), no statistically significant differences were found between the clear zones formed by L. plantarum Ln4 and LGG.
We determined the MICs of Lactobacillus strains and LGG against the oral pathogenic bacterium S. mutans KCTC 5124.L. plantarum Ln4, L. plantarum NK181, and LGG inhibited S. mutans KCTC 5124 growth at concentrations of 12.5%, 6.25%, and 12.5%, respectively.LGG was used as the positive control strain compared with Lactobacillus strains (data not shown).

Cell Aggregation and Cell Surface Hydrophobicity
The effects of Lactobacillus strains on auto-aggregation, co-aggregation, and cell surface hydrophobicity of S. mutans KCTC 5124 were shown in Table 2.After 4 h of incubation, S. mutans KCTC 5124 showed a low autoaggregation value (18.23%); however, it increased after 24 h of incubation (70.99%).
In addition, the cell surface hydrophobicity was measured by bacterial adhesion to hydrocarbons, when compared to the control and treated Lactobacillus strains [25,26].The cell surface hydrophobicity of L. plantarum strains against S. mutans KCTC 5124 was associated with its adhesion ability, as shown in Fig. 1.The control group (S.mutans KCTC 5124), which is untreated with Lactobacillus strains, showed 23.60% cell surface hydrophobicity.However, L. plantarum Ln4 (16.75%) and L. plantarum NK181 (16.56%) treated groups had significantly reduced cell surface hydrophobicity values (p < 0.05).

Total EPS Production Rate
When the total EPS production rate of S. mutans KCTC 5124 was studied, the results indicated that Lactobacillus strains led to a reduction in the total EPS production by S. mutans KCTC 5124 (Fig. 2).Among the Lactobacillus strains, L. plantarum Ln4 significantly reduced the total EPS production (34.98%), and following, LGG, L. rhamnosus  L. plantarum NK181 also showed an inhibitory effect on EPS production (18.85%).These results were significantly different from those of the control (untreated Lactobacillus stains) (p < 0.001).
CLSM was used to evaluate biofilm formation inhibition by L. plantarum Ln4 (Fig. 4).S. mutans KCTC 5124 and LGG were used as negative and positive controls, respectively.Compared to that of negative control, biofilm formation was reduced in Lactobacillus-strains and LGG-were treated groups.Among them, S. mutans KCTC 5124 treated with L. plantarum Ln4 showed the highest biofilm inhibition.

Discussion
Dental caries is known as a major disease related with oral condition, which is multi-species biofilm-mediated [16].It has been previously reported that probiotics promote oral health.Specifically, S. mutans is a major oral pathogenic bacteria associated with dental caries, and antimicrobial activity plays a vital role suppressing these  dental caries [27].The biofilm formed by S. mutans secretes glucosyltransferases that synthesize glucans to promote bacterial binding (adhesion) to the tooth surface.Adhesion is thus critical for biofilm progression [28].Biofilms are formed by microbial communities to resist a variety of conditions and to protect bacterial cells by attaching tenaciously to each other [5,29].Consequently, control of early step is important to inhibit biofilmformation by S. mutans [5].The biofilm can be suppressed by antimicrobial activity, which could be affected by organic acids, hydrogen peroxide, bacteriocin, and biosurfactants [7,30].This study evaluated the antimicrobial effect of L. plantarum stains against S. mutans KCTC 5124.Our findings showed that L. plantarum antimicrobial and antibiofilm activities inhibited biofilm formation by S. mutans KCTC 5124.Among the tested Lactobacillus strains, L. plantarum Ln4 showed the highest antimicrobial activity against S. mutans KCTC 5124 using the deferred method and MIC test.In particular, compared to L. plantarum 200661, L. plantarum Ln4 showed higher antibacterial activity at the same concentration [7].Consequently, we evaluated that L. plantarum Ln 4 could use as potential strain when compared to Weissella cibaria CMU and Lactobacillus reuteri DSM 17938 (widely recognized oral probiotics) [31,32].
Generally, autoaggregation, cell surface hydrophobicity, and EPS production are related to bacterial adhesion to the tooth surface and are important elements to consider when aiming to prevent biofilm formation by S. mutans [16].It has been proven that bacteria can better colonize the tooth surface when they exhibit high hydrophobic activity.Especially, biofilm formed by S. mutans related with sucrose and hydrophobic activity results in attached to tooth surface [26].
In addition, EPS are the major factor in forming, maturing, maintaining, and expending the S. mutans biofilm matrix.Thus, we investigated the effects of autoaggregation, hydrophobicity, and EPS production on L. plantarum Ln4 against S. mutans.We determined that L. plantarum Ln4 had the greatest effects of aggregation, hydrophobic activity, and EPS production changes among the tested Lactobacillus strains.Moreover, regarding cell surface properties, L. plantarum Ln4 significantly reduced EPS production by S. mutans (p < 0.001).Taken together, results suggest that L. plantarum Ln4 might be expected to prevent cavity by inhibiting the aggregation of S. mutans.
A previous study reported that coaggregation of Lactobacillus sp.strains with S. mutans ATCC 25175 varied between 6.32% to 20.93%.In addition, compared to the autoaggregation of S. mutans ATCC 25175, the coaggregation of S. mutans ATCC 25175 with L. plantarum sp.strains was low [31].Another study demonstrated that L. plantarum K25 decreased EPS formation (21.44%) as well as the antimicrobial peptide GH12, which has a dental caries effect at 1/4 and 1/2 MIC, remarkably reduced EPS [33].
We also conduct to CLSM analysis to measure the bacterial counts as staining cells in biofilm [34].CLSM as microscopy methods confirmed inhibition of biofilm by S. mutans in treated Lactobacillus strains.Among the strains, L. plantarum Ln4 showed the highest biofilm formation inhibition (Fig. 4).In general, it is difficult to remove a mature biofilm than early biofilm and then biofilm degradation is vital to measure the antibiofilm activity against S. mutans.Because biofilm protect oral bacteria and S. mutans return cellular damage by stress through induce membrane protein [16].One study reported that L. plantarum FB-T9 inhibited biofilm formation by S. mutans depending on the incubation time, when compared to the control [35].In addition, another study demonstrated that Lactobacillus strains inhibit biofilm formation by S. mutans [19].L. brevis KCCM reported that the highest antibiofilm effect against S. mutans KCTC 5458 at MIC levels [16].It has been reported that coaggregation with lactic acid bacteria and reduces EPS production by physical interference.
This study demonstrated that L. plantarum Ln4 has potential effects to prevent dental caries using its antimicrobial activity and to be applied as food additives in oral health industry.

Conclusion
The oral health effects of probiotics have recently been investigated.Among the oral pathogenic bacteria, S. mutans is major bacteria that influences dental caries.L. plantarum Ln4 was previously evaluated for probiotic characteristics and other functional activities.In this study, our findings demonstrated the antimicrobial and antibiofilm activities of L. plantarum Ln4 against the oral pathogenic bacterium S. mutans KCTC 5124 through autoaggregation, coaggregation, cell surface hydrophobicity, EPS production rate, and inhibition of biofilm formation analyses.L. plantarum Ln4 was effective when compared with the control, which was not treated with Lactobacillus strains, in all experiments.Therefore, L. plantarum Ln4 could inhibit biofilm formation of oral pathogenic bacteria and is expected to be used in the healthcare industry.